Editing Gibson Assembly (section)

== Enhancement ==

5x Isothermal Reaction Mix was created by combining 3 ml 1 M Tris-HCl, pH = 7.5 (ThermoFisher Scientific 15567027), 300 µl 1 M MgCl2 (Millipore Sigma M1028), 600 µl 10 mM dNTP mix (Goldbio D-900-1), 46.3 mg DTT (Goldbio DTT10), 20 mg NAD (Millipore Sigma N0632), 1.5 g PEG&nbsp;&nbsp;8000 (Millipore Sigma 89510), and brought to 6 ml with UltraPure water (ThermoFisher Scientific 10977015). 320 µl aliquots were stored at -80C. To create a 1.33x Gibson Assembly mix, 40 units Phusion High-Fidelity DNA Polymerase (NEB M0530), 12 units T5 exonuclease (NEB M0363), and 6,400 units Tth DNA Ligase (homemade, supplementary protocol 1) were added to 320 ul of 5x Isothermal Reaction Mix. For the enhanced formulation, <span style="color:#FF0000;">'''10 ul ET SSB (500 µg/ml, NEB M2401S)'''</span>was also added. UltraPure water (ThermoFisher Scientific 10977015) was added to bring the final volume of each to 1.2 ml. Gibson Assembly reaction mixes were dispensed into single use aliquots and stored at -20C.&nbsp;

Assembly Reactions All reactions included each insert fragment at a 6-fold molar excess to the vector fragment. Each 10 µl assembly reaction included 4 fmol of vector along with 24 fmol of each insert fragment and 0.4 fmol of an intact supercoiled kanamycin-resistant plasmid (as a transformation control). For both the original and enhanced Gibson Assembly formulations, 2.5 µl of this DNA mixture was added to 7.5 µl of 1.33x master mix on ice. For the NEBuilder® HiFi reactions, 2.5 µl of this DNA mixture and 2.5 µl of UltraPure water was added to 5 µl of NEBuilder® HiFi DNA Assembly Master Mix (NEB E2621) on ice. After mixing, the reactions were moved directly to 50C for one hour and then placed back on ice. Both the 2-insert and 6-insert assemblies were run in triplicate for each reaction formulation.

Transformation 15 µl of UltraPure water was added to each assembly reaction on ice and 2.5 µl of these diluted reactions were then transformed into 50 µl chemically competent DH5α cells (prepared in house, Zymo T3001) on ice. Following a five minute incubation on ice, 450 µl of pre-warmed SOC outgrowth medium (NEB B9020S) was added and the transformation was incubated at 37C with shaking (250 RPM) for one hour. 200 µl of each transformation was then plated on an LB-agar plate with carbenicillin (100 µg/ml) and another 200 µl was plated on an LB-agar plate with kanamycin (50 µg/ml). Plates were incubated overnight at 37C Compared to the original Gibson Assembly formulation, this enhanced formulation exhibited markedly increased accuracy for the 6-insert assembly (>80% compared to ~20%, p = 3e-5), and greatly increased efficiency in both 2-insert and 6-insert assemblies (>12-fold increase, p &lt; 0.05). Compared to the NEBuilder® HiFi DNA Assembly Master Mix (NEB E2621), the enhanced formulation showed a trend toward higher assembly efficiency, but this trend failed to reach statistical significance. However, the enhanced formulation showed significantly increased accuracy in the 6-insert assembly (&gt;80% compared to ~45%, p = 0.00024). These results clearly demonstrate that the addition of ET SSB can increase the efficiency and accuracy of Gibson Assembly, especially as the number of fragments being assembled increases. While the exact mechanism has yet to be conclusively elucidated, it is possible that this ssDNA binding protein may facilitate specific hybridization and protect ssDNA overhangs from endonuclease attack.4, 6 The simplicity of this modification makes it easy to use in place of the original formulation without any other modifications. Furthermore, by making this mix in-house with homemade Thermus thermophilus DNA Ligase, this mix can be made for less than $10 per milliliter, making it accessible in a wide variety of research settings.&nbsp;



Homemade Assembly Mixes Both the original and enhanced Gibson Assembly mixes were based on recipes from OpenWetWare. 9 Briefly, a 5x Isothermal Reaction Mix was created by combining 3 ml 1 M Tris-HCl, pH = 7.5 (ThermoFisher Scientific 15567027), 300 µl 1 M MgCl2 (Millipore Sigma M1028), 600 µl 10 mM dNTP mix (Goldbio D-900-1), 46.3 mg DTT (Goldbio DTT10), 20 mg NAD (Millipore Sigma N0632), 1.5 g PEG&nbsp;8000 (Millipore Sigma 89510), and brought to 6 ml with UltraPure water (ThermoFisher Scientific 10977015). 320 µl aliquots were stored at -80C. To create a 1.33x Gibson Assembly mix, 40 units Phusion High-Fidelity DNA Polymerase (NEB M0530), 12 units T5 exonuclease (NEB M0363), and 6,400 units Tth DNA Ligase (homemade, supplementary protocol 1) were added to 320 ul of 5x Isothermal Reaction Mix. For the enhanced formulation, 10 ul ET SSB (500 µg/ml, NEB M2401S) was also added. UltraPure water (ThermoFisher Scientific 10977015) was added to bring the final volume of each to 1.2 ml. Gibson Assembly reaction mixes were dispensed into single use aliquots and stored at -20C.&nbsp;